While astrocytes establish the largest syncytium in the brain through gap junctions, the basic role of syncytial coupling in astrocyte function remains poorly understood. This project takes a novel approach by considering syncytial coupled astrocytes as a functional system based on a mechanism we?ve just discovered. We show that astrocytes are strongly electrically coupled to achieve syncytial isopotentiality, which enables astrocytes to be more efficient in potassium clearance. Based on this, our long-term goal is to understand the mechanisms by which an astrocyte syncytium functions as an integral part of neural circuitries. A central hypothesis of this proposal is that syncytial isopotentiality requires a developmentally mature anatomic structure in a syncytium and can be dynamically regulated by physiological neuronal activity. Because syncytial isopotentiality approaches maturity after the postnatal day 12, the first objective is to determine the anatomic basis underpinning syncytial isopotentiality. We will follow postnatal development to identify the anatomic features underpinning CA1 syncytial isopotentiality. The 3-D structure of CA1 syncytium will be resolved by CLARITY in ALDHIL1-eGFP transgenic mice using confocal microscopy. The ultra-structural details of process-to-process contacts and gap junctions between neighboring astrocytes will be resolved by blockface serial scanning EM. Mathematical models will be used to biophysically rationalize the resultant anatomic database that explains syncytial isopotentiality. The second objective is to identify mechanisms that coordinate dynamic neuronal activity, coupling strength, and isopotentiality. We hypothesize that, in order to maintain syncytial isopotentiality, intensive neuronal activity increases the gating probability of gap junctions through a Ca2+-dependent mechanism. Double patch recording from astrocytes in hippocampal slices and pairs of freshly dissociated astrocytes will be used in proposed studies. The third objective is to determine the pathological impact of epileptic neuronal discharge and structurally altered syncytia in disease models on syncytial isopotentiality. We hypothesize that syncytial isopotentiality can be acutely disrupted by epileptic neuronal firing and chronically disrupted by disease induced astrogliosis. Epileptic neuronal firings will be induced by picrotoxin/Mg2+-free bath solution applied to slices. Astrogliosis, induced by cuprizone in CA1 and by amyloid precursor protein (APP) overexpression in the dentate gyrus, will be used to create structurally altered syncytia over the course of astrogliosis progression. The expected outcomes from the proposed works are to establish a clear syncytial anatomy and isopotentiality relationship, to prove that under physiological conditions, the astrocyte syncytium and its associated neuronal circuitries are interactive components in the brain, and to corroborate above notions from disease model studies. Such results are expected to shed new lights for a novel research direction, in which the mysterious astrocyte function in the adult brain can be explored at a higher hierarchy, syncytial system level, in the normal brain as well as in neurological disorders.